Immune stimulating macrolides

ABSTRACT

The present invention provides immune stimulating macrolides of formula (I), wherein the substituents are as defined in claim  1 . The macrolides have utility in treating intracellular bacterial, fungal, and protozoal infections.

FIELD OF THE INVENTION

The present invention provides novel macrolide compounds capable ofstimulating the immune system. The present invention relates to novelcompounds for use in medicine, notably in the treatment of intracellularbacterial, fungal, and protozoal infections and in the co-treatment ofviral disease, chronic inflammatory conditions, and cancer whenstimulation of the immune system is beneficial. The compounds may alsobe used as immune modulating adjuvants in vaccination. The novelmacrolides maximize the modulating effects of the immune system whileminimizing the therapeutically unwanted direct antibacterial effects.The present invention also provides methods for preparing the compoundsof the invention and for use of the compounds in medicine.

BACKGROUND OF THE INVENTION

Intracellular bacterial, fungal, and protozoal infections are often notdiagnosed in healthy individuals as they appear asymptomatic, or becausethe symptoms are mild enough that the infected individual is notinclined to seek medical assistance. As such, intracellular infectionsmay persist latently or may progress to a disease state. Conditionsinterfering with normal T cell function usually leads to progression ofthe disease from a latent infection, and intracellular infections suchas Mycobacterium tuberculosis (Mtb) are a common cause of death inpatients where HIV infection has progressed to AIDS. There is thus agreat need in the art for methods and means of treating intracellularinfections.

Intracellular pathogens such as Mtb have the capacity to hide withinintracellular compartments in monocytes and macrophages causingpersistent infections. Although Mtb are recognized by CD4⁺ T helpercells in the lung and an appropriate response is mounted, the systemfails to create sterilizing immunity (MacMicking 2012). To escape immunerecognition by the host, Mtb have developed a series of mechanism thatinhibits recognition of Mtb peptides presented in the MHC class IIpocket for CD4⁺ T helper cells. Toll like receptor 2 has beendemonstrated to be inhibited by Mtb, which in turn inhibits IFN-γinduced MHC class II expression (Noss 2001). In addition, data suggestthat Mtb has the capacity to inhibit phagosome processing andmaturation, possibly by an invariant chain associated mechanism(Ramachandra 2001). Therefore, the normal antigen processing, loadingand presentation of MHC class II peptides derived from Mtb is impaireddue to Mtb produced immune escape factors.

The endosomal lysosomal pathway is designed to take up pathogens,process them into 12-15 aa long peptides, peptides, that after theremoval of the Invariant chain peptide CLIP by HLA-DM, are loaded intothe MHC class II pocket. The antigen loading is followed by transport ofthe MHC class II-peptide complex to the cell surface for presentationfor the specific T cell receptor of CD4⁺ T helper cells (Roche 2015).Recently the Mtb expressed protein EsxH has been reported to directlyinhibit the endosomal sorting complex required for transport (ESCRT)machinery (Portal-Celhay 2016). EsxH inhibits the ability of antigenpresenting monocytes and macrophages to activate CD4⁺ T helper cells.Since intact ESCRT machinery seems necessary for antigen processing,presentation and activation of T cells, EsxH is the link that explainsMtb induced immune escape by intervening with the MHC class II pathway.

The importance of MHC class II presentation has also been demonstratedin patients with primary immunodeficiencies (PID). PID patients withdefects in the IFN-γ circuit, involving IFNGR and IL-12, have anincreased risk of acquiring TBC and atypical mycobacterial infections.Since MHC class II expression is dependent upon and regulated by IFN-γexpression, defects in the IFN-γ circuit will result in additionallydecreased MHC class II expression and a poor activation of CD4⁺ T helpercells.

Protozoa such as Toxoplasma gondii have developed a mechanism to avoidimmune recognition by hiding intracellularly as an obligateintracellular parasite. The mechanism involves interference with MHCclass II expression and thus diminishes the amount of Toxoplasma gondiito be presented for specific CD4⁺ T helper cells. The detailed mechanismis dependent on soluble proteins expressed by Toxoplasma gondii thatinhibit IFN-gamma induced expression of MHC class II (Leroux 2015).

Furthermore, it has been demonstrated that different fungal infectionsare dependent on MHC class II expression. Cryptococos neoformans maycause life threatening brain infections in patients withimmunodeficiencies including HIV. Work in a mouse model of Cryptococosneoformans infection has demonstrated that the activation of microglialcells and their upregulation of MHC class II, in an IFN-gamma dependentmanner, is critical for survival (Zhou 2007).

Therefore, to overcome the immune escaping mechanisms induced by Mtb andother intracellular bacteria, protozoa such as Toxoplasma gondii, orfungi exemplified by Cryptococcus, an increased expression of MHC classII on the cell surface of monocytes, macrophages, microglia, or otherinfected cells is likely beneficial for immune recognition andelimination of the pathogen.

Macrolides, such as erythromycin and azithromycin, have been used foryears in the treatment of bacterial infections. Erythromycin is apolyketide natural product macrolide produced by fermentation of theactinomycete Saccharopolyspora erythraea. Azithromycin is asemisynthetic azalide derivative of erythromycin. Many references existdescribing the antibacterial activity of macrolides, such aserythromycin. This antibacterial mechanism is achieved through moleculebinding to the P-site on the bacterial 50S bacterial ribosome, thusinterfering with the tRNA binding.

Many references describe generation of analogues of erythromycin viasemisynthesis and biosynthetic engineering. In particular, methods havebeen described for semisynthetic removal of the glycosyl groups onerythromycin, desosamine and mycarose/cladinose. Further methods havebeen described for biotransformation to add alternative glycosyl groupsto the erythromycin aglycone (eg see Gaisser et al., 2000, Schell etal., 2008 and WO2001079520). The main focus of this published work,however, has been to generate antibacterial erythromycin analogues.

DESCRIPTION OF THE INVENTION

Immune stimulating activity from macrolides that lack directantibacterial activity has previously not been reported. Surprisingly,we found that compounds of the invention, such as compound 1 (FIG. 1),had a potent immune stimulating effect on several cell types of theimmune system. After 24-48 h of in vitro stimulation of peripheral bloodmononuclear cells (PBMC) with 1 μM compound 1, the activation markerCD69 was upregulated on CD4 T cells and B cells (FIG. 2). We alsoobserved upregulation of the MHC class I molecule (HLA-ABC) on T- andB-cells (FIG. 3), indicating an effect on antigen presentation ofantigens derived from intracellular infections. Stimulation of monocytesin the PBMC population with compound 1 led to the upregulation of theco-stimulatory molecule CD80 as well as the antigen presenting moleculeMHC class II (HLA-DR) (FIG. 4). Monocytes differentiated intomacrophages also upregulated CD80 in response to stimulation by compound1 (FIG. 5). Furthermore, PBMCs stimulated with compound 1 expressed analtered cytokine profile with increased production of theimmunosuppressive cytokine IL-10, indicating an immune inhibitory effectunder certain conditions. Further analysis of the immunological effectof compound 1 revealed an altered cytokine driven proliferation profileof T cells after six days stimulation, measured with flow cytometry(FIG. 7). In addition, virus specific T cell proliferation was affectedby compound 1. PBMCs from cytomegalovirus (CMV) infected donors culturedin the presence of CMV antigen and compound 1 displayed an alteredphenotype of activated CMV specific CD8+ T cells with an increasedexpression of IL-7 receptor α (CD127) (FIG. 8). CD127 is crucial for Tcell homeostasis, differentiation and function, and reduced expressioncorrelates with disease severity in HIV and other chronic viral diseases(Crawley et al. 2012).

In summary, compound 1 has a surprising ability to specifically activateand modify an immune response by affecting antigen presentation,co-stimulation and T cell activation and proliferation. In many of thesestudies, compound 2 (FIG. 1), another related macrolide erythromycinanalogue with altered glycosylation, previously published in Schell etal. 2008 (as compound 20), was included as negative control since itshowed little or no activity in the assays.

Thus, in one aspect of the invention, there is provided immunestimulating macrolides of Formula (I) (also referred to as “compounds ofthe invention” or “compounds of Formula (I)”) or a pharmaceuticallyacceptable salt, hydrate, solvate, tautomer, enantiomer or diastereomerthereof:

wherein X is selected from C═O, —NR₃CH₂—, —CH₂NR₃—, —NR₃(C═O)—,—(C═O)NR₃—, C═NOH, and —CH(OH)—, and R₂ is a sugar of Formula (II) orFormula (III):

wherein R₁ is selected from an alkyl, heteroalkyl, cycloalkyl, aryl, andheteroaryl moiety, wherein alkyl moiety is selected from C₁-C₆ alkylgroups that are optionally branched, wherein heteroalkyl moiety isselected from C₁-C₆ alkyl groups that are optionally branched orsubstituted and that optionally comprise one or more heteroatoms,wherein cycloalkyl moiety is selected from a C₁-C₆ cyclic alkyl groupsthat are optionally substituted and that optionally comprise one or moreheteroatoms,wherein aryl moiety is selected from optionally substituted C₆ aromaticrings,wherein heteroaryl moiety is selected from optionally substituted C₁-C₅aromatic rings comprising one or more heteroatoms,wherein heteroatoms are selected from O, N, P, and S,wherein substituents, independently, are selected from alkyl, OH, F, Cl,NH₂, NH-alkyl, NH-acyl, S-alkyl, S-acyl, O-alkyl, and O-acyl,wherein acyl is selected from C₁-C₄ optionally branched acyl groups,wherein R₃ is selected from H and Me,wherein R₄ is selected from H and Me,wherein R_(a) is selected from H and CR₂₁R₂₂R₂₃,wherein R₂₁, R₂₂, R₂₃, and R₅, R₆, R₇, R₈, R₉, and R₁₀, independently,are selected from H, Me, NR₁₁R₁₂, NO₂, and OR₁₁,wherein R₂₃ together with R₄ in Formula (II), R₄ together with R₅ inFormula (II), R₅ together with R₇ in Formula (II), and R₇ together withR₉ in Formula (II), independently, may be joined to represent a bond toleave a double bond between the carbon atoms that each group isconnected to, so thatwherein if R₂₃ and R₄ are joined to form a double bond, then Formula(II) can be represented by:

wherein if R₄ and R₅ are joined to form a double bond, then Formula (II)can be represented by:

wherein if R₅ and R₇ are joined to form a double bond, then Formula (II)can be represented by:

wherein if R₇ and R₉ are joined to form a double bond, then Formula (II)can be represented by:

wherein R₄ together with R₅ in Formula (III), R₄ together with R₇ inFormula (III), and R₇ together with R₉ in Formula (III), independently,may be joined to represent a bond to leave a double bond between thecarbon atoms that each group is connected to, so thatwherein if R₄ and R₅ are joined to form a double bond, then Formula(III) can be represented by:

wherein if R₄ and R₇ are joined to form a double bond, then Formula(III) can be represented by:

wherein if R₇ and R₉ are joined to form a double bond, then Formula(III) can be represented by:

wherein R₂₁ together with R₂₂, R₅ together with R₆, R₇ together with R₈,or R₉ together with R₁₀ may be replaced with a carbonyl,wherein R₁₁ and R₁₂, independently, are selected from H and alkyl,wherein R₁₃ is selected from H, OH, and OCH₃,wherein R₁₄ is selected from H and OH,and wherein one of R₅, R₆, R₇, R₈, R₉ or R₁₀ is selected from NR₁₁R₁₂and NO₂,with the proviso that when R₁ is Et, R₂ is a sugar of Formula (II), R₁₃is H or OH, R₁₄ is H or OH, R_(a) is H, R₄ is Me, R₅ is H, R₆ is OH, R₇is H, R₈ is NR₁₁R₁₂, R₉ is H, and R₁₀ is H, X may not be C═O.with the proviso that when R₁ is Et, R₂ is a sugar of Formula (II), R₁₃is H or OH, R₁₄ is H or OH, R_(a) is H, R₄ is Me, R₅ is OH, R₆ is H, R₇is OH, R₈ is Me, R₉ is H, and R₁₀ is H, X may not be C═O.with the proviso that when R₁ is Et, R₂ is a sugar of Formula (II), R₁₃is H or OH, R₁₄ is H or OH, R_(a) is H, R₄ is Me, R₅ is OH, R₆ is H, R₇is H, R₈ is NR₁₁R₁₂, R₉ is H, and R₁₀ is OH, X may not be C═O.

In another aspect of the invention, there is provided immune stimulatingmacrolides of Formula (I: or a pharmaceutically acceptable salt,hydrate, solvate, tautomer, enantiomer or diastereomer thereof

wherein X is selected from C═O, —NR₃CH₂—, and —CH(OH)—, and R₂ is asugar of Formula (II):

wherein R₁ is selected from and alkyl or cycloalkyl moiety,wherein alkyl moiety is selected from C₁-C₆ alkyl groups that areoptionally branched and, independently, optionally hydroxylated,wherein cycloalkyl moiety is selected from C₁-C₆ optionally substitutedcyclic alkyl groups,wherein substituents are selected from alkyl and OH,wherein R₃ is selected from H and Me,wherein R₄ is selected from H and Me,wherein R_(a) is selected from H and CR₂₁R₂₂R₂₃,wherein R₂₁, R₂₂, R₂₃, and R₅, R₆, R₇, R₈, R₉, and R₁₀, independently,are selected from H, Me, NR₁₁R₁₂, NO₂, and OR₁₁,wherein R₂₃ together with R₄ in Formula (II), R₄ together with R₅ inFormula (II), R₅ together with R₇ in Formula (II), and R₇ together withR₉ in Formula (II), independently, may be joined to represent a bond toleave a double bond between the carbon atoms that each group isconnected to, so thatwherein if R₂₃ and R₄ are joined to forma double bond, then Formula (II)can be represented by:

wherein if R₄ and R₅ are joined to form a double bond, then Formula (II)can be represented by:

wherein if R₅ and R₇ are joined to form a double bond, then Formula (II)can be represented by:

wherein if R₇ and R₉ are joined to form a double bond, then Formula (II)can be represented by:

wherein R₂₁ together with R₂₂, R₅ together with R₆, R₇ together with R₈,or R₉ together with R₁₀ may be replaced with a carbonyl,wherein R₁₁ and R₁₂, independently, are selected from H and alkyl,wherein R₃ is selected from H, OH, and OCH₃,wherein R₁₄ is selected from H and OH,and wherein one of R₅, R₆, R₇, R₈, R₉ or R₁₀ is selected from NR₁₁R₁₂and NO₂, with the proviso that when R₁ is Et, R₂ is a sugar of Formula(II), R₁₃ is H or OH, R₁₄ is H or OH, R_(a) is H, R₄ is Me, R₅ is H, R₆is OH, R₇ is H, R₈ is NR₁₁R₁₂, R₉ is H, and R₁₀ is H, X may not be C═O.with the proviso that when R₁ is Et, R₂ is a sugar of Formula (II), R₁₃is H or OH, R₁₄ is H or OH, R_(a) is H, R₄ is Me, R₅ is OH, R₆ is H, R₇is OH, R₈ is Me, R₉ is H, and R₁₀ is H, X may not be C═O.with the proviso that when R₁ is Et, R₂ is a sugar of Formula (II), R₁₃is H or OH, R₁₄ is H or OH, R_(a) is H, R₄ is Me, R₅ is OH, R₆ is H, R₇is H, R₈ is NR₁₁R₁₂, R₉ is H, and R₁₀ is OH, X may not be C═O.

In another aspect of the invention, there is provided a method forproducing a compound of formula (I), which involves addition of anaglycone with formula IV to a culture of a biotransformation strainwhich glycosylates at the 3-hydroxyl position.

In a preferred embodiment of this aspect of the invention, thebiotransformation strain expresses glycosyltransferases with 70% or morehomology to AngMII (SEQ ID no. 1) or AngMIII (SEQ ID no. 2), such aswith 75% or more, with 80% or more, with 90% or more or with 95% or morehomology such as 100% homology.

The homology between two amino acid sequences or between two nucleicacid sequences is described by the parameter “identity”. Alignments ofsequences and calculation of homology scores may be done using e.g. afull Smith-Waterman alignment, useful for both protein and DNAalignments. The default scoring matrices BLOSUM50 and the identitymatrix are used for protein and DNA alignments respectively. The penaltyfor the first residue in a gap is −12 for proteins and −16 for DNA,while the penalty for additional residues in a gap is −2 for proteinsand −4 for DNA. Alignment may be made with the FASTA package versionv20u6. Multiple alignments of protein sequences may be made using“ClustalW”. Multiple alignments of DNA sequences may be done using theprotein alignment as a template, replacing the amino acids with thecorresponding codon from the DNA sequence. Alternatively, differentsoftware can be used for aligning amino acid sequences and DNAsequences. The alignment of two amino acid sequences is e.g. determinedby using the Needle program from the EMBOSS package (http://emboss.org)version 2.8.0. The substitution matrix used is BLOSUM62, gap openingpenalty is 10, and gap extension penalty is 0.5.

An interesting selection of compounds of the invention are compoundswherein R₂ is selected from L-daunosamine, L-acosamine, L-ristosamine,D-ristosamine, 4-oxo-L-vancosamine, L-vancosamine, D-forosamine,L-actinosamine, 3-epi-L-vancosamine, L-vicenisamine, L-mycosamine,D-mycosamine, D-3-N-methyl-4-O-methyl-L-ristosamine, D-desosamine,N,N-dimethyl-L-pyrrolosamine, L-megosamine, L-nogalamine, L-rhodosamine,D-angolosamine, L-kedarosamine, 2′-N-methyl-D-fucosamine,3-N,N-dimethyl-L-eremosamine, D-ravidosamine,3-N,N-dimethyl-D-mycosamine/D-mycaminose, 3-N-acetyl-D-ravidosamine,4-O-acetyl-D-ravidosamine, 3-N-acetyl-4-O-acetyl-D-ravidosamine,D-glucosamine, N-acetyl-D-glucosamine, L-desosamine, D-amosamine,D-viosamine, L-avidinosamine, D-gulosamine, D-allosamine, andL-sibirosamine.

Yet another interesting selection of compounds of the invention arecompounds wherein R₂ is selected from D-angolosamine, N-desmethylD-angolosamine, N-didesmethyl D-angolosamine, N-desmethyl N-ethylD-angolosamine, and N-didesmethyl N-diethyl D-angolosamine.

Yet another interesting selection of compounds of the invention arecompounds wherein R₂ is selected from N-desmethyl D-angolosamine,N-didesmethyl D-angolosamine, N-desmethyl N-ethyl D-angolosamine, andN-didesmethyl N-diethyl D-angolosamine.

Yet another interesting selection of compounds of the invention iscompounds wherein R₂ is a sugar according to Formula (II).

Yet another interesting selection of compounds of the invention arecompounds wherein R₂ is a sugar according to formula (II) wherein R_(a)is H, R₄ is Me, R₅ is H, R₆ is OH, R₇ is H, R₈ is NR₁₁R₁₂, R₉ is H andR₁₀ is H.

Yet another interesting selection of compounds of the invention arecompounds wherein R₁₁ is selected from H, Me, and Et, and R₁₂ isselected from H, Me, and Et.

Yet another interesting selection of compounds of the invention arecompounds wherein R₁₁ is Et and R₁₂ is Et.

Yet another interesting selection of compounds of the invention arecompounds wherein R₁₁ is Me and R₁₂ is Et.

Yet another interesting selection of compounds of the invention iscompounds wherein X is selected from C═O, —NR₃CH₂— and —CH(OH)—

Yet another interesting selection of compounds of the invention iscompounds wherein R₁ is selected from Me, Et, and cycloalkyl.

Yet another interesting selection of compounds of the invention arecompounds wherein R₁ is selected from Me and Et.

Yet another interesting selection of compounds of the invention arecompounds wherein X is selected from —NR₃CH₂— or —CH₂NR₃—.

Yet another interesting selection of compounds of the invention arecompounds wherein one of R₅, R₆, R₇, or R₈, is NR₁₁R₁₂.

Yet another interesting selection of compounds of the invention arecompounds wherein R₂₁, R₂₂, R₂₃, and R₅, R₆, R₇, R₈, R₉, and R₁₀,independently, are selected from H, Me, NR₁₁R₁₂, and OR₁₁,

Yet another interesting selection of compounds of the invention arecompounds wherein R₁₃ and R₁₄ are OH.

Of particular interest are compounds of Formula (I), wherein R₁ is Et,R₂ is a sugar of Formula (II), R₁₃ is OH, R₁₄ is H, R_(a) is H, R₄ isMe, R₅ is H, R₆ is OH, R₇ is H, R₈ is NR₁₁R₁₂, R₉ is H, R₁₀ is H, and Xis C═O.

Specific compounds according to the invention include:

As seen from the examples herein some of the compounds of the inventionare without substantial antibacterial activity as defined herein.

General Chemistry Methods

The skilled person will recognise that the compounds of the inventionmay be prepared, in known manner, in a variety of ways. The routes beloware merely illustrative of some methods that can be employed for thesynthesis of compounds of Formula (I).

Where an aglycone is required for biotransformation these can beaccessed in a number of ways. Azithromycin and erythromycin are readilyavailable and considered suitable starting points. Themycarose/cladinose and/or desosamine are removed by chemical methods,such as glycoside cleavage. Briefly, in one method the sugars may beremoved by treatment with acid. In order to facilitate removal of theamino sugar it is first necessary to oxidise the dimethylamine to forman N-oxide which is then removed by pyrolysis. The resulting 5-O/3-Osugars can then be removed by acidic degradation. A suitable method istaught by LeMahieu et al. 1974 and Djokic et al. 1988. Finally, thecompound is biotransformed using a bacterial strain which adds the aminosugar.

Another route to suitable aglycones is by fermentation and isolationfrom a suitable blocked mutant. For example, erythronolide B (3a) can begenerated by fermentation of strains of S. erythraea blocked inglycosylation, such as strains and processes described, for example, inU.S. Pat. No. 3,127,315 (e.g. NRRL2361, NRRL2360, NRRL2359 andNRRL2338), Gaisser et al. 2000 (e.g. S. erythraea DM ΔBV ΔCIII).Briefly, the fermentation is conducted by methods known in the art.Typically, a seed culture is prepared and transferred to a productionvessel. The production phase is between 4 and 10 days and the organismis grown between 24° C. and 30° C. with suitable agitation and aeration.The aglycone can then be isolated by extraction and purification.

Where an aglycone or compound of the invention possesses an amino sugaror any other tertiary amine and is prepared by fermentation, it will benecessary to extract the bacterial broth and purify the compound.Typically, the bacterial broth is adjusted to between pH 8 and 10,ideally 9.5. The broth can then be extracted with a suitable organicsolvent. This solvent not be water miscible and is ideally ethylacetate, methyl tert-butyl ether (MTBE) or solvents with similarproperties. The broth and the solvent are mixed, ideally by stirring,for a period of time, e.g. 30 minutes or 1 hour. The phases are thenseparated and the organic extracts removed. The broth can be extractedin this manner multiple times, ideally two or three times. The combinedorganic extracts can then be reduced in vacuo. The residue is thendissolved or suspended in mildly acidic aqueous solvent. Typically, thisis an ammonium chloride aqueous solution. This is then extracted with awater-immiscible organic solvent, such as ethyl acetate, a number oftimes, ideally 2 or 3 times. The resulting aqueous layer is collectedand the pH is adjusted to between pH 8 and 10, ideally 9.0. Theresultant aqueous layer is then extracted with a water-immiscibleorganic solvent, such as ethyl acetate, a number of times, ideally 2 or3 times. The organic extracts are combined and reduced in vacuo to yielda crude extract enhanced in the target compound requiring furtherpurification. Compound purification can be done by chromatography or(re)crystallisation, and the methods required are well known to a personskilled in the art. Where chromatography is required on normal phasesilica and an aglycone or compound of the invention possesses an aminosugar or other tertiary amine, then it is beneficial to add a basicmodifier to the mobile phase. For instance, chromatography on normalphase silica can use a hexane, ethyl acetate, methanol system forelution with 0-5% aqueous ammonium hydroxide added. Ideally, 2% aqueousammonium hydroxide is added. Following biotransformation, both unusedaglycone and compound of the invention can be purified separately fromthe same crude extract using a suitable solvent system. If furtherpurification is required, this may optionally be carried out bypreparative HPLC.

Reductive amination to alkylate a primary or secondary amine is wellknown to a person skilled in the art. The amine is mixed in a solventwith an aldehyde or ketone and a reducing agent is added. Sodiumborohydride can then reduce the imine or hemiaminal that results fromthe reaction of the amine and carbonyl, resulting in e.g. an alkylatedamine. Sodium borohydride may also reduce other carbonyl groups present,e.g. ketones. In cases where a ketone also exists, it is preferred touse a reducing agent that is more specific to a protonated imine, suchas sodium cyanoborohydride, though it will be obvious to a personskilled in the art that different reducing agents, solvents,temperatures, and reaction times may need to be tested to find theoptimal conditions.

General Use of the Compounds of the Invention

Compounds of the invention as described herein can be used in medicine,medical research or in the manufacture of a composition for such use.Accordingly, when in the following the term “compounds of the invention”is used in connection with medical use or pharmaceutical composition,the term is intended also to include the compounds of Formula (I)provided that such compounds have not been known for such a use. Inparticular, medical use as described herein of the compounds of Formula(I) includes compounds, wherein when R₁ is Et, R₂ is a sugar of Formula(II), R₁₃ is OH, R₁₄ is H, R_(a) is H, R₄ is Me, R₅ is H, R₆ is OH, R₇is H, R₈ is NR₁₁R₁₂, R₉ is H, R₁₀ is H, and X is C═O.

The compounds of the invention are designed in order to minimize directantibacterial effects, but rather focus on immune activating properties.When a compound of the invention is added to cultures of bacteria E.coli, S. salivarius, L. casei, B. longum or M. luteus, no or minimalantibacterial effect is recognized. The advantage of having compoundswith isolated immune stimulatory properties that effect the host cellsis that development of bacterial resistance is avoided. In addition, thewell-known side effect of macrolides affecting the gut microbiota, withthe risk of overgrowth of Clostridium difficile causing diarrhea andpseudomebraneous colitis, is avoided. Many intracellular pathogens suche.g. Mtb, as well as viruses and cancers have developed mechanisms toavoid immune recognition, i.e. by down regulating HLA expression, toavoid detection by T cells. The mechanism of the compounds of theintervention rely on the activation and increased expression of HLAmolecules on infected cells. HLA molecules load and present peptidesderived from intracellular infectious agents in order to present arecognition signal for T cells allowing elimination of infected cells.

The compounds of the invention disclosed herein may be used in thetreatment of intracellular bacterial, fungal, and protozoal infections,such as bacterial infections caused by Mycobacterium tuberculosis,Mycobacteria causing atypical disease, Mycobacterium avium and M.intracellulare (also known as Mycobacterium avium-intracellularecomplex, or MAC), M. kansasii, M. marinum, M. fortuitum, M. gordinae,Mycoplasma pneumoniae, M. genitalium, M. hominis, Ureaplasmaurealyticum, U. parvum, Chlamydophila pneumoniae, and Salmonellatyphimurium, such as protozoal infections caused by Toxoplasma gondii,Plasmodium falciparum, P. vivax, Trypanosoma cruzi, Cryptosporidium, andLeishmania, and such as fungal infections caused by Histoplasmacapsulatum, Cryptococcus neoformans, and Encephalitozoon cuniculi.

The compounds of the invention may be used treatment of intracellularbacterial, fungal, and protozoal infections when these infections occuralone or in association with viral agents or viral disease or inassociation with other causes of primary or secondary immunodeficiency.Causes of primary immunodeficiency include inherited geneticdeficiencies and somatic mutations, whereas secondary immunodeficiencymay be caused by viral infections such as those described above, or byinheritable or non-inheritable conditions such as diabetes mellitus, ormalnutrition, or by agents such as immunodepressants, drug abuse, orother environmental factors.

Moreover, the compounds of the invention disclosed herein may be used asa treatment or co-treatment for diseases, disorders, conditions, andsymptoms, where immune response stimulation is useful, such as intreating patients infected with viral agents or with viral diseases suchas HIV, Adenovirus, Alphavirus, Arbovirus, Borna Disease, Bunyavirus,Calicivirus, Condyloma Acuminata, Coronavirus, Coxsackievirus,Cytomegalovirus, Dengue fever virus, Contageous Ecthyma, Epstein-Barrvirus, Erythema Infectiosum, Hantavirus, Viral Hemorrhagic Fever, ViralHepatitis, Herpes Simplex Virus, Herpes Zoster virus, InfectiousMononucleosis, Influenza, Lassa Fever virus, Measles, Mumps, MolluscumContagiosum, Paramyxovirus, Phlebotomus fever, Polyoma-virus, RiftValley Fever, Rubella, Slow Disease Virus, Smallpox, Subacute SclerosingPanencephalitis, Tumor Virus Infections, West Nile Virus, Yellow FeverVirus, Rabies Virus, and Respiratory Syncitial Virus.

Moreover, the compounds of the invention are contemplated to be suitablefor use in the co-treatment of cancer, in particular Adrenal Cancer,Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNSTumors, Breast Cancer, Castleman Disease, Cervical Cancer, Colon/RectumCancer, Endometrial Cancer, Esophagus Cancer, Eye Cancer, GallbladderCancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal StromalTumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, KaposiSarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, AcuteMyeloid Leukemia, Chronic Lymphocytic Leukemia, Acute LymphocyticLeukemia, Chronic Myeloid Leukemia, Chronic Myelomonocytic Leukemia,Liver Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, LungCarcinoid Tumor, Lymphoma, Malignant Mesothelioma, Multiple Myeloma,Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer,Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavityand Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, PancreaticCancer, Penile Cancer, Pituitary Tumors, Prostate Cancer,Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Basal andSquamous Cell Skin Cancer, Melanoma, Merkel Cell Skin Cancer, SmallIntestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer,Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer,Waldenstrom Macroglobulinemia, and Wilms Tumor.

Thus, the advantageous properties of the compound of the invention overthe prior art macrolides may include one or more of the following:

-   -   Reduced direct antibacterial activity    -   Improved MHC class I stimulation    -   Improved immunomodulation    -   Improved activation of antigen presenting cells    -   Improved T-cell response    -   Improved antiviral activity    -   Improved MHC class II antigen presentation

Pharmaceutical Compositions Comprising a Compound of the Invention

The present invention also provides a pharmaceutical compositioncomprising a compound of the invention together with one or morepharmaceutically acceptable diluents or carriers. Similarly, the presentinvention also provides a pharmaceutical kit comprising at least onepharmaceutical composition comprising a compound of the inventiontogether with one or more pharmaceutically acceptable excipients. Thepresent invention also relates to cosmetic or veterinary compositionscomprising a compound of the invention together with one or morecosmetically or veterinary acceptable excipients.

The compounds of the invention or pharmaceutical, cosmetic, orveterinary compositions comprising compounds of the invention may beadministered by any conventional route such as but not limited toparenteral, oral, topical, or via a mucosa (including buccal,sublingual, transdermal, vaginal, rectal, nasal, ocular, etc.), via amedical device (e.g. a stent), or by inhalation. The treatment mayconsist of a single administration or a plurality of administrationsover a period of time.

The dosage regimen of the compounds of the invention and thepharmaceutical compositions of the invention may be varied depending onthe pharmaceutical properties of the compound or composition inquestion. The dosage regimen may consist of a single administration or aplurality of administrations over one or more periods of time.Administration may be once daily, twice daily, three times daily, fourtimes daily, less frequently, or more frequently, depending on thespecific use, the disease to be treated, and the physical condition andcharacteristics (such as gender, weight, and age) of the patient to betreated. The treatment may also be by continuous administration such ase.g. intravenous administration via a drop or via depots orsustained-release formulations.

Whilst it is possible for a compound of the invention to be administeredas such, it is preferable to present it as a pharmaceutical compositiontogether with one or more pharmaceutically acceptable excipients. Theexcipient(s) must be “acceptable” in the sense of being compatible withthe compound of the invention and not deleterious to the recipientsthereof. Examples of suitable excipients are described in more detailbelow.

The pharmaceutical compositions may conveniently be presented in asuitable dosage form including a unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. Such methodsinclude the step of bringing into association the compound of theinvention with one or more excipients. In general, the pharmaceuticalcompositions are prepared by uniformly and intimately bringing intoassociation the compound of the invention with the excipient(s), andthen, if necessary, shaping the resulting composition into e.g. atablet.

A compound of the invention will normally be administered by anyconventional administration route, such as the oral or any parenteralroute, in the form of a pharmaceutical composition comprising thecompound of the invention, optionally in the form of a pharmaceuticallyacceptable salt, in a pharmaceutically acceptable dosage form. Dependingupon the disorder and patient to be treated, as well as the route ofadministration, the pharmaceutical composition may be administered atvarying doses and/or frequencies.

The pharmaceutical compositions must be stable under the conditions ofmanufacture and storage; thus, if necessary, they should be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. In case of liquid formulations such as solutions, dispersion,emulsions, and suspensions, the excipient(s) can be a solvent ordispersion medium such as but not limited to water, ethanol, polyol(e.g. glycerol, propylene glycol and liquid polyethylene glycol),vegetable oils, and suitable mixtures thereof, as well as a solvent ordispersion medium comprising water, ethanol, polyol (e.g. glycerol,propylene glycol and liquid polyethylene glycol), and vegetable oils.

For example, the compounds of the invention may be administered orally,buccally or sublingually in the form of tablets, capsules, films,ovules, elixirs, solutions, emulsions, or suspensions, which may containflavouring or colouring agents.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of a compoundof the invention; as multiple units e.g. in the form of a tablet orcapsule; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The pharmaceuticalcompositions may also be presented as a bolus, electuary, or paste.

Solutions or suspensions of the compounds of the invention suitable fororal administration may also contain one or more solvents includingwater, alcohol, polyol, etc., as well as one or more excipients such aspH-adjusting agents, stabilizing agents, surfactants, solubilizers,dispersing agents, preservatives, flavours, etc. Specific examplesinclude N,N-dimethylacetamide, polysorbate 80, polyethylene glycol, andPhosal 50 PG (which consists of phosphatidylcholine, soya-fatty acids,ethanol, mono/diglycerides, propylene glycol and ascorbyl palmitate).The pharmaceutical compositions of the present invention may also be inthe form of emulsions, wherein a compound according to Formula (I) maybe presented in an emulsion such as an oil-in-water emulsion or awater-in-oil emulsion. The oil may be a natural or synthetic oil or anyoil-like substance such as e.g. soy bean oil or safflower oil orcombinations thereof.

Tablets may contain excipients such as microcrystalline cellulose,lactose (e.g. lactose monohydrate or anhydrous lactose), sodium citrate,calcium carbonate, dibasic calcium phosphate and glycine, butylatedhydroxytoluene (E321), crospovidone, hypromellose, disintegrants such asstarch (preferably corn, potato or tapioca starch), sodium starchglycollate, croscarmellose sodium, and certain complex silicates, andgranulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),macrogol 8000, sucrose, gelatin, and acacia. Additionally, lubricatingagents such as magnesium stearate, stearic acid, glyceryl behenate, andtalc may be included.

A tablet may be made by compression or moulding of a compounds of theinvention, optionally with one or more excipients. Compressed tabletsmay be prepared by compressing in a suitable machine the compound of theinvention in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g. povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g. sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose), surface-active agent,and/or dispersing agent. Moulded tablets may be made by moulding in asuitable machine a mixture of the powdered compound of the inventionmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be further treated of processed to provide slowor controlled release of the compound of the invention contained thereinusing, for example, hydroxypropylmethylcellulose in varying proportionsto provide desired release profile.

Solid pharmaceutical compositions of a similar type may also be employedas fillers in gelatin capsules. Preferred excipients in this regardinclude lactose, starch, cellulose, milk sugar, and high molecularweight polyethylene glycols. For aqueous suspensions and/or elixirs, thecompounds of the invention may be combined with various sweetening orflavouring agents, colouring matter or dyes, with emulsifying and/orsuspending agents, and with diluents such as water, ethanol, propyleneglycol and glycerin, and combinations thereof.

Pharmaceutical compositions of the invention suitable for topicaladministration in the oral cavity include lozenges comprising a compoundof the invention in a flavoured basis, usually sucrose and acacia ortragacanth; pastilles comprising a compound of the invention in an inertbasis such as gelatin and glycerin, or sucrose and acacia; andmouth-washes comprising a compound of the invention in a suitable liquidcarrier.

Pharmaceutical compositions of the invention adapted for topicaladministration may be prepared as ointments, creams, suspensions,lotions, powders, solutions, pastes, gels, impregnated dressings,sprays, aerosols or oils, transdermal devices, dusting powders, and thelike. Such compositions may be prepared via conventional methodscontaining a compound of the invention. Thus, they may also comprisecompatible excipients, such as preservatives, solvents to assist drugpenetration, emollient in creams or ointments, and ethanol or oleylalcohol in lotions. Excipients may constitute from about 1% w/w to about98% w/w of the composition. Preferably, excipients constitute up toabout 80% w/w of the composition. As an illustration only, a cream orointment is prepared by mixing sufficient quantities of hydrophilicmaterial and water, containing from about 5-10% w/w of the compound, insufficient quantities to produce a cream or ointment having the desiredconsistency.

Pharmaceutical compositions of the invention adapted for transdermaladministration may be presented as discrete patches intended to remainin intimate contact with the epidermis of the recipient for a prolongedperiod of time. For example, a compound of the invention may bedelivered from the patch by iontophoresis.

For applications to external tissues, for example the mouth and skin,the pharmaceutical compositions of the invention are preferably appliedas a topical ointment or cream. When formulated in an ointment, acompound of the invention may be employed with either a paraffinic or awater-miscible ointment base.

Alternatively, a compound of the invention may be formulated in a creamwith an oil-in-water cream base or a water-in-oil base.

For parenteral administration, fluid unit dosage forms are preparedcomprising a compound of the invention and a sterile vehicle, such asbut not limited to water, alcohols, polyols, glycerine, and vegetableoils, with water being preferred. The compound of the invention,depending on the vehicle and concentration used, can be eithercolloidal, suspended, or dissolved in the vehicle. In preparingsolutions, the compound of the invention can be dissolved in water forinjection and filter sterilised before filling into a suitable vial orampoule and sealing.

Advantageously, agents such as local anaesthetics, preservatives, andbuffering agents can be dissolved in the vehicle. To enhance thestability, the pharmaceutical composition can be frozen after fillinginto the vial, and the water may then be removed under vacuum. The drylyophilized powder is then sealed in the vial and an accompanying vialof water for injection may be supplied to reconstitute the liquid priorto use.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, such pharmaceutical compositions can be in the form ofsterile powders for the extemporaneous preparation of such sterileinjectable solutions or dispersions. In all cases, the final injectableform must be sterile and must be effectively fluid for easysyringability.

Parenteral suspensions are prepared in substantially the same manner assolutions, except that the compound of the invention is suspended in thevehicle instead of being dissolved, and sterilization cannot beaccomplished by filtration. The compound of invention can be sterilisedby exposure to ethylene oxide before suspending in the sterile vehicle.Advantageously, a surfactant or wetting agent is included in thepharmaceutical composition to facilitate uniform distribution of thecompound of the invention.

It should be understood that in addition to the ingredients particularlymentioned above, the pharmaceutical compositions of the presentinvention may include other agents conventional in the art having regardto the type of formulation in question. For example, thosepharmaceutical compositions suitable for oral administration may includeflavouring agents. A person skilled in the art will know how to choose asuitable formulation and how to prepare it, e.g. with guidance fromRemington's Pharmaceutical Sciences, 18^(th) edition, Mack PublishingCompany, 1990, or a newer edition. A person skilled in the art will alsoknow how to choose a suitable administration route and dosage.

It will be recognized by a person skilled in the art that the optimalquantity and spacing of individual dosages of a compound of theinvention will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration, and the ageand condition of the particular subject being treated, and that aphysician will ultimately determine appropriate dosages to be used. Thisdosage may be repeated as often as appropriate. If side effects develop,the amount and/or frequency of the dosage can be altered or reduced, inaccordance with normal clinical practice.

All % values mentioned herein are % w/w unless the context requiresotherwise.

Definitions

The articles “a”, “an”, and “the” are used herein to refer to one or tomore than one (i.e. at least one) of the grammatical objects of thearticle. By way of example “an analogue” means one analogue or more thanone analogue.

As used herein the terms “compound(s) of the invention” and “compoundsof Formula (I)” are used interchangeably and refer to compounds ofFormula (I).

As used herein the term “direct antibacterial effect” refers to theantibacterial activity of erythromycin and analogues which occursthrough binding to the bacterial rRNA complex. This effect does notrequire presence of any host immune system components and therefore isapparent in standard antibacterial assays such as in vitro MinimumInhibitory Concentration (MIC) assays and disk inhibition assays.

As used herein the term “without substantial antibacterial activity” isintended to mean that the compound of the invention has a MIC valueof >64 μg/ml when tested in accordance with Example 13 herein for itsantibacterial activity in E. coli, S. salivarius, L. casei and B.longum.

As used herein the term “alkyl” refers to any straight or branched chaincomposed of only sp3-hybridized carbon atoms, fully saturated withhydrogen atoms such as e.g. —C_(n)H_(2n+1) for straight chain alkyls,wherein n can be in the range of 1 and 6 such as e.g. methyl, ethyl,propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,neopentyl, isopentyl, hexyl or isohexyl. The alkyl as used herein may befurther substituted.

The term “heteroalkyl” in the present context designates a group—X—C—₁₋₆ alkyl used alone or in combination, wherein C₁₋₆ alkyl is asdefined above and X is O, S, NH or N-alkyl. Examples of linearheteroalkyl groups are methoxy, ethoxy, propoxy, butoxy, pentoxy andhexoxy. Examples of branched heteroalkyl are iso-propoxy, sec-butoxy,tert-butoxy, iso-pentoxy and iso-hexoxy. Examples of cyclic heteroalkylare cyclopropyloxy, cyclobutyloxy, cyclopentyloxy and cyclohexyloxy. Theheteroalkyl as used herein may be further substituted.

As used herein the term “cycloalkyl” refers to a cyclic/ring structuredcarbon chains having the general formula of —C_(n)H_(2n−1) where n isbetween 3-6, such as e.g. cyclopropyl, cyclobytyl, cyclopentyl orcyclohexyl and the like. The cycloalkyl as used herein may be furthersubstituted or contain a heteroatom (O, S, NH or N-alkyl) in the cyclicstructure.

The term “aryl” as used herein is intended to include carbocyclicaromatic ring systems. Aryl is also intended to include the partiallyhydrogenated derivatives of the carbocyclic systems enumerated below.

The term “heteroaryl” as used herein includes heterocyclic unsaturatedring systems containing one or more heteroatoms selected among nitrogen,oxygen and sulphur, such as furyl, thienyl, pyrrolyl, and is alsointended to include the partially hydrogenated derivatives of theheterocyclic systems enumerated below.

The terms “aryl” and “heteroaryl” as used herein refers to an aryl,which can be optionally unsubstituted or mono-, di- or tri substituted,or a heteroaryl, which can be optionally unsubstituted or mono-, di- ortri substituted. Examples of “aryl” and “heteroaryl” include, but arenot limited to, phenyl, biphenyl, indenyl, naphthyl (1-naphthyl,2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl,N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl,3-anthracenyl), phenanthrenyl, fluorenyl, pentalenyl, azulenyl,biphenylenyl, thiophenyl (1-thienyl, 2-thienyl), furyl (1-furyl,2-furyl), furanyl, thiophenyl, isoxazolyl, isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, pyranyl, pyridazinyl, pyrazinyl,1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl,benzofuranyl, benzothiophenyl (thianaphthenyl), indolyl, oxadiazolyl,isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl,acridinyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl,quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, phteridinyl,azepinyl, diazepinyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl),5-thiophene-2-yl-2H-pyrazol-3-yl, imidazolyl (1-imidazolyl,2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl(1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl, 1,2,3-triazol-4-yl,1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl),thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl,3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl), isoquinolyl (1-isoquinolyl,3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl,7-isoquinolyl, 8-isoquinolyl), quinolyl (2-quinolyl, 3-quinolyl,4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl),benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl,4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl,7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl(2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl),4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl),6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl)),benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl,4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl,7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl(2-(2,3-dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl),4-(2,3-dihydro-benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl),6-(2,3-dihydro-benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl)),indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl,6-indolyl, 7-indolyl), indazolyl (1-indazolyl, 2-indazolyl, 3-indazolyl,4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl,(1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl,6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl(1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl,2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl,7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl,4-carbazolyl). Non-limiting examples of partially hydrogenatedderivatives are 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl,pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyland the like.

The pharmaceutically acceptable salts of the compound of the inventioninclude conventional salts formed from pharmaceutically acceptableinorganic or organic acids or bases as well as quaternary ammonium acidaddition salts. More specific examples of suitable acid salts includehydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric,fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic,tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, toluenesulfonic, methanesulfonic,naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic,malic, steroic, tannic and the like. Other acids such as oxalic, whilenot in themselves pharmaceutically acceptable, may be useful in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable salts. Morespecific examples of suitable basic salts include sodium, lithium,potassium, magnesium, aluminium, calcium, zinc,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine and procaine salts.

LEGENDS TO FIGURES

FIG. 1. The structures of the macrolides Erythromycin A, Compound 1,Compound A, compound B and EM703.

FIG. 2. CD69 upregulation on T- and B-cells. PBMC were treated for 24 hwith compound 1, compound A and activation controls LPS and IFN-gamma.The expression of the early activation marker CD69 was measured on theCD4+ T cell population (left) and CD19+ B cell population (right) withflow cytometry. Values represents mean fluorescent intensity, MFI, anderror bars standard deviation in the triplicate samples.

FIG. 3. HLA-A,B,C upregulation on T- and B-cells. PBMC were treated for24 h with compounds 1 or A and activation controls LPS and IFN-γ. Theexpression of HLA-A,B,C was measured on the CD4+ T cell population(left) and CD19+ B cell population (right) with flow cytometry. Valuesrepresents mean fluorescent intensity, MFI, and error bars standarddeviation in the triplicate samples.

FIG. 4. CD80 and HLA-DR upregulation on blood monocytes. PBMC weretreated for 24 h with compounds 1 or A as well as activation controlsLPS and IFN-gamma. The expression of CD80 and HLA-DR was measured on themonocyte cell population with flow cytometry. Values represents meanfluorescent intensity, MFI, and error bars standard deviation in thetriplicate samples.

FIG. 5. CD80 upregulation on blood monocytes. PBMC were treated for 24 hwith compounds 1 or A as well as activation control IFN-gamma. Theexpression of CD80 was measured on the monocyte cell population withflow cytometry. Values represents mean fluorescent intensity, MFI, anderror bars standard deviation in the triplicate samples.

FIG. 6. Production of IL-10 from PBMCs after stimulation with compound 1for 48 h or 1 week, measured with ELISA.

FIG. 7. CD4 T cell proliferation after 6 days stimulation with compound1, measured with proliferation dye Celltrace violet (Invitrogen) andflow cytometry. Untreated cells (UNT) or compound A were used ascontrols.

FIG. 8. Upregulation of IL-7 receptor α (CD127) on CMV specific CD8 Tcells after incubation with compound 1, measured with flow cytometry.

FIG. 9: Interferon-gamma secretion (as measured by cytometric beadassay) from PBMCs (from a CMV+ donor) grown with CMV peptides in thepresence or absence of compound 1 or A for 5 days.

FIG. 10: Interferon-gamma secretion (as measured by cytometric beadassay) from macrophages stimulated with indicated compound for 48 h.

FIG. 11: Chemokine RANTES secretion (as measured by cytometric beadassay) from PBMC or macrophages stimulated with indicated compound for48 h.

FIG. 12: IL12p70 secretion (as measured by cytometric bead assay) fromPBMC or macrophages stimulated with indicated compound for 48 h.

FIG. 13: IL1b secretion (as measured by cytometric bead assay) fromPBMC, macrophages or CD4 T cells stimulated with indicated compound for48 h.

FIG. 14: % CD25 high cells in blood of C57bl/6 mice injected 24 hpreviously with indicated dose of compound 1. CD25 expression wasmeasured by flow cytometry.

FIG. 15: % MHC class I high CD11b+ cells in spleen of 3 individualC57bl/6 mice injected 24 h previously with indicated compound. MHC classI and CD11 b expression was measured by flow cytometry.

EXPERIMENTAL

Materials

Unless otherwise indicated, all reagents used in the examples below areobtained from commercial sources. Example suppliers of Azithromycin Binclude Santa Cruz Biotechnology (Texas, USA) and Toronto ResearchChemicals (Toronto, Canada).

Antibodies

Anti-CD80 V450, anti-CD69 PE, anti HLA-DR APC-R700, anti CD127-APC, andanti-Anti-HLA-A,B,C FITC were purchased from BD Biosciences. Celltraceviolet for T cell proliferation assay was purchased from Invitrogen.ELISA antibodies were purchased from BD Biosciences.

Media

RPMI-1640 (Invitrogen) supplemented with 25 mM HEPES, L-glutamine,Sodium pyruvate, 10% fetal bovine serum (Gibco), 100 μg/mL penicillinand 100 μg/mL streptomycin

General Biology Methods

The effect of the compounds of the invention on immune stimulation maybe tested using one or more of the methods described below:

General Compound Methods

Compound Analysis—Solubility and Stability in Solution

Analysis of Fermentation Broths and Compounds

An aliquot of fermentation broth obtained as described below was shakenvigorously for 30 minutes with an equal volume of ethyl acetate, andthen separated by centrifugation, or the already isolated compounds weredissolved in methanol:water (9:1, 0.1 mg/ml), and then separated bycentrifugation. Supernatants were analysed by LC-MS and LC-MS/MS andchromatography was achieved over base-deactivated Luna C18reversed-phase silica (5 micron particle size) using a Luna HPLC column(250×4.6 mm; Phenomenex (Macclesfield, UK)) heated at 40° C. Agilent1100 HPLC system comprising of quaternary pump, auto sampler, columnoven and diode array detector coupled to a Bruker Esquire ion trap MS.

Mobile phase A=0.1% formic acid in water

Mobile phase B=0.1% formic acid in acetonitrile

Gradient: T=0 min, B=50%; T=4.5 min, B=50%; T=7 min, B=100%; T=10.5 min,B=100%; T=10.75 min, B=50%; T=13 min, B=50%.

Compounds were identified by LC-MS and LC-MS/MS and quantified byLC-MS/MS against an internal standard.

Analysis of Marker Expression by Flow Cytometry

Human peripheral blood mononuclear cells (PBMCs) were purified fromhealthy donors with Ficoll-Paque density centrifugation. Cells werecultured in complete RPMI-1640 media (Invitrogen) supplemented with 25mM HEPES, L-glutamine, Sodium pyruvate (Sigma), 10% fetal bovine serum,100 μg/mL penicillin and 100 μg/mL streptomycin (Hyclone) for 24-72hours in 37° C., 5% CO₂ and stimulated with and increasingconcentrations of compound 1 and 2. Cells were then washed in PBS andstained with monoclonal antibodies specific for cell surface markers (BDPharmingen) and analysed with flow cytomtetry using a BD FACS Canto IIflow cytometer. All samples were tested in duplicates.

Cytomegalovirus (CMV) Cultures

Human peripheral blood mononuclear cells (PBMCs) were purified fromhealthy CMV positive donors with Ficoll-Paque density centrifugation.The PBMC were labeled with 5 μM celltrace violet (Invitrogen) in PBS for15 minutes and then washed with complete cell culture medium. Thelabeled PBMC was cultured in the presence of a peptide library spanningthe CMV pp65 protein (1 μg peptide/ml, JPT) in AIM-V media (Invitrogen)supplemented with L-glutamine, Sodium pyruvate (Sigma), 10% fetal bovineserum, 100 μg/mL penicillin and 100 μg/mL streptomycin (Hyclone) for 6-8days in 37° C., 5% CO₂. Cell proliferation was assessed with flowcytomtery using a BD FACS Canto II flow cytometer.

ELISA

Supernatant IL-10 was measured with a standard sandwich ELISA (allantibodies from BD Biosciences) after 48 hours and 7 days incubationwith 2.5 μM of compound 1 and 100 U/mL IL-2 (Miltenyi Biotechnologies)in complete RPMI media, 37° C., 5% CO₂

TLR2 Assay

Samples and controls were tested in duplicate on recombinant HEK-293-TLRcell lines using a cell reporter assay at Invivogen using their standardassay conditions. These cell lines functionally over-express human TLR2protein as well as a reporter gene which is a secreted alkalinephosphatase (SEAP). The production of this reporter gene is driven by anNFkB inducible promoter. The TLR reporter cell lines activation resultsare given as optical density values (OD). 20 μl of each test articlewere used to stimulate the hTLR2 reporter cell lines in a 200 μl offinal reaction volume. Samples were tested in duplicate, with at leasttwo concentrations tested—20 uM and 10 uM.

Assessment of Cell Permeability (Bidirectional)

10 μM Test article was added to the apical (A) surface of Caco-2 cellmonolayers (in HBSS buffer with 0.3% DMSO and 5 μM LY at 37 degrees C.)and compound permeation into the basolateral (B) compartment measuredfollowing 90 minutes incubation. This was also performed in the reversedirection (basolateral to apical) to investigate active transport.LC-MS/MS is used to quantify levels of both the test and standardcontrol compounds. Efflux ratio was calculated by dividing the B to Apermeability by the A to B permeability.Drug permeability:Papp=(VA/(Area×time))×([drug]accepter/(([drug]initial,donor)×DilutionFactor)

Assessment of Metabolic Stability (Microsome Stability Assay)

Rate of metabolism in microsomes was tested as follows:

Human liver microsomes were diluted with buffer C (0.1 M PotassiumPhosphate buffer, 1.0 mM EDTA, pH 7.4) to a concentration of 2.5 mg/mL.Microsomal stability studies were carried out by adding 30 μL of 1.5 μMcompound spiking solution to wells (1.5 μL of 500 μM spiking solution(10 μL of 10 mM DMSO stock solution into 190 μL ACN to eventuallygenerate final test concentration of 1 uM) and 18.75 μL of 20 mg/mLliver microsomes into 479.75 μL of Buffer C). All samples werepre-incubated for approximately 15 minutes at 37° C. Following this, thereaction was initiated by adding 15 μL of the NADPH solution (6 mM) withgentle mixing. Aliquots (40 μL) were removed at 0, 5, 15, 30 and 45minutes and quenched with ACN containing internal standard (135 μL).Protein was removed by centrifugation (4000 rpm, 15 min) and the sampleplate analysed for compound concentration by LC-MS/MS. Half-lives werethen calculated by standard methods, comparing the concentration ofanalyte with the amount originally present.

EXAMPLES Example 1—Preparation of Compound 1

Preparation of Azithromycin Aglycone (Az-AG) (1a)

Azithromycin aglycone (1a) was generated using methods described in theliterature (Djokic et al. 1988). In brief, azithromycin is converted toazithromycin aglycone by the acidic removal of the 3-O and 5-O sugars.The 5-O amino sugar is first oxidised and pyrolyzed to facilitatecleavage.

Generation of Biotransformation Strains Capable of GlycosylatingErythromycin Aglycones (Erythronolides):

Generation of S. erythraea 18A1 (pAES52)

pAES52, an expression plasmid containing angAI, angAII, angCVI,ang-orf14, angMIII, angB, angMI and angMII along with the actII-ORF4pactI/III expression system (Rowe et al., 1998) was generated asfollows.

The angolamycin sugar biosynthetic genes were amplified from a cosmidlibrary of strain S. eurythermus ATCC23956 obtained from the AmericanType Culture Collection (Manassas, Va., USA). The biosynthetic genecluster sequence was deposited as EU038272, EU220288 and EU232693(Schell et al. 2008).

The biosynthetic gene cassette was assembled in the vector pSG144 asdescribed previously (Schell et al. 2008, ESI), adding sequential genesuntil the 8 required for sugar biosynthesis were obtained, creatingplasmid pAES52.

pAES52 was transformed into strain 18A1 (WO2005054265).

Transformation of pAES52 into S. erythraea 18A1

pAES52 was transformed by protoplast into S. erythraea 18A1 usingstandard methods (Kieser et al. 2000, Gaisser et al. 1997). Theresulting strain was designated ISOM-4522, which is deposited at theNCIMB on 24 Jan. 2017 with Accession number: NCIMB 42718.

Generation of S. erythraea SGT2 (pAES54)

pAES54, an expression plasmid containing angAI, angAII, angCVI,ang-orf14, angMIII, angB, angMI and angMII along with the actII-ORF4pactI/Ill expression system (Rowe et al., 1998) was generated as follows

The angolamycin sugar biosynthetic genes were amplified from a cosmidlibrary of strain S. eurythermus ATCC23956 obtained from the AmericanType Culture Collection (Manassas, Va., USA). The biosynthetic genecluster sequence was deposited as EU038272, EU220288 and EU232693(Schell, 2008).

The biosynthetic gene cassette was assembled in the vector pSG144 asdescribed previously (Schell, 2008, ESI), adding sequential genes untilthe 8 required for sugar biosynthesis were obtained, creating plasmidpAES52.

Plasmid pAES54 was made by ligating the 11,541 bp Spel-Nhel fragmentcontaining the actII-ORF4 pactI/Ill promotor system and the 8 ang geneswas excised from pAES52 with the 5,087 bp Xbal-Spel fragment from pGP9,containing an apramycin resistance gene, oriC, oriT for transfer instreptomycetes and phiBT1 integrase with attP site for integrativetransformation. (The compatible Nhel and Xbal sites were eliminatedduring the ligation.)

pAES54 was then transformed into S. erythraea SGT2 (Gaisser et al. 2000,WO2005054265).

Transformation of pAES54 into S. erythraea SGT2

pAES54 was transferred by conjugation into S. erythraea SGT2 usingstandard methods. In brief, E. coli ET12567 pUZ8002 was transformed withpAES54 via standard procedures and spread onto 2TY with Apramycin (50μg/mL), Kanamycin (50 μg/mL), and Chloramphenicol (33 μg/mL) selection.This plate was incubated at 37° C. overnight. Colonies from this wereused to set up fresh liquid 2TY cultures which were incubated at 37° C.until late log phase was reached. Cells were harvested, washed, mixedwith spores of S. erythraea SGT2, spread onto plates of R6 and incubatedat 28° C. After 24 hours, these plates were overlaid with 1 mL ofsterile water containing 3 mg apramycin and 2.5 mg nalidixic acid andincubated at 28° C. for a further 5-7 days. Exconjugants on this platewere transferred to fresh plates of R6 containing apramycin (100 μg/mL).

Alternative Biotransformation Strain

Alternatively, BIOT-2945 (Schell et al. 2008) may be used as thebiotransformation strain, as this also adds angolosamine toerythronolides.

Biotransformation of Azithromycin Aglycone

Erlenmeyer flasks (250 mL) containing SV2 medium (40 mL) and 8 uLthiostrepton (25 mg/mL) were inoculated with 0.2 mL of spore stock ofstrain ISOM-4522 and incubated at 30° C. and shaken at 300 rpm with a2.5 cm throw for 48 hours.

SV2 media: Ingredient Amount glycerol 15 g glucose 15 g soy peptone A3SC15 g NaCl  3 g CaCO₃  1 g RO water To final volume of 1 LPre-sterilisation pH adjusted to pH 7.0 with 10M HCl Sterilised byautoclaving @ 121° C., 30 minutes

Sterile bunged falcon tubes (50 mL) containing EryPP medium (7 mL) wereprepared and inoculated with culture from seed flask (0.5 mL per falcontube) without antibiotics. The falcons were incubated at 30° C. andshaken at 300 rpm with a 2.5 cm throw for 24 hours.

ERYPP medium: Ingredient Amount toasted soy flour (Nutrisoy) 30 gglucose 50 g (NH₄)₂SO₄  3 g NaCl  5 g CaCO₃  6 g RO water To finalvolume of 1 L Pre-sterilisation pH adjusted to pH 7.0 with 10M HClSterilised in situ by autoclaving @ 121° C., 30 minutes Poststerilisation 10 ml/L propan-1-ol added

After 24 hours, azithromycin aglycone (0.5 mM in DMSO, 50 uL) was addedto each falcon tube and incubation continued at 300 rpm with a 2.5 cmthrow for a further 6 days.

Isolation of Compound 1

Whole broth was adjusted to pH 9.5 and extracted twice with one volumeof ethyl acetate. The organic layers were collected by aspirationfollowing centrifugation (3,500 rpm, 25 minutes). The organic layerswere combined and reduced in vacuo to reveal a brown gum that containedcompound 1. This extract was partitioned between ethyl acetate (200 ml)and aqueous ammonium chloride (20 ml of a 50% concentrated solution).After separation, the organic layer was extracted with a further volume(200 ml) of the ammonium chloride aqueous solution. The combined aqueouslayers were then adjusted to pH 9.0 with aqueous sodium hydroxide andthen extracted twice with one volume equivalent of ethyl acetate. Theorganic layers were combined and reduced in vacuo to a brown solid. Thisextract was then applied to a silica column and eluted step wise (in 500ml lots) with:

Solvent Hexanes EtOAc MeOH Aq. NH₄OH A 0.499 0.499 0 0.002 B 0.250 0.7480 0.002 C 0 0.998 0 0.002 D 0 0.988 0.01 0.002 E 0 0.978 0.02 0.002 F 00.968 0.03 0.002 G 0 0.958 0.04 0.002

Compound 1 was predominantly in F and G. These solvents were combinedand reduced in vacuo to yield a brown solid containing compound 1. Thismaterial was then purified by preparative HPLC (C18 Gemini NX column,Phenomenex with 20 mM ammonium acetate and acetonitrile as solvent).Fraction containing the target compound were pooled and taken to drynessfollowed by desalting on a C18 SPE cartridge.

Example 2—Preparation of Compound 3 (Known Compound—Corresponds toCompound 17 in Schell et al., 2008)

Erythronolide B (3a) can be generated by fermentation of strains of S.erythraea blocked in glycosylation, such as strains and processesdescribed, for example, in U.S. Pat. No. 3,127,315 (e.g. NRRL2361, 2360,2359 and 2338), Gaisser et al 2000 (e.g. S. erythraea DM ΔBV ΔCIII.

Erythronolide B (3a) was then fed to a biotransformation strain capableof adding angolosamine to the 3-hydroxyl (such as NCIMB 42718) andcompound 3 was isolated from the fermentation broth by standard methods.

Example 3—Preparation of Compound 4

Azithromycin B aglycone (4a) was generated by hydrolysis of the sugarsfrom azithromycin B in the same way as for azithromycin A.

Azithromycin B aglycone (4a) was then fed to a biotransformation straincapable of adding angolosamine to the 3-hydroxyl (such as NCIMB 42718)and isolated from the fermentation broth using standard methods.

Example 4—Preparation of Compound 5

Cyclobutyl erythronolide B (5a) was generated using methods described inWO98/01571. In brief, S. erythraea DM ΔBV ΔCIII (Gaisser et al. 2000)was transformed with pIG1 (Long et al., 2002, WO98/01571). Fermentationof the resulting strain with addition of cyclobutene carboxylic acid ledto production of Cyclobutyl erythronolide B (5a). This was isolated fromfermentation broths using standard methods. Cyclobutyl erythronolide B(5a) was then fed to a biotransformation strain capable of addingangolosamine to the 3-hydroxyl (such as NCIMB 42718) and compound 5isolated from the fermentation broth using standard methods.

Example 5—Preparation of Compound 6

A methyl group was removed from the aminosugar of compound 3 (seeexample 2) by adding it to a fermentation of ATCC 31771 and isolatingcompound 6 from the fermentation broth using standard methods.

Example 6—Preparation of Compound 7

Compound 3 was treated with sodium borohydride in solvent. Followingstandard reaction work up compound 7 was purified by standard methods.

Example 7—Preparation of Compound 8

14-desmethyl erythronolide B (8a) was generated using methods describedin WO2000/00618. In brief, S. erythraea DM ΔBV ΔCIII (Gaisser et al.2000) was transformed with pPFL43. The resulting strain was fermentedusing typical methods and compound 8a was isolated using chromatography.

14-desmethyl erythronolide B (8a) was then fed to a biotransformationstrain capable of adding angolosamine to the 3-hydroxyl (such as NCIMB42718) and isolated from the fermentation broth using standard methods.

Example 8—Preparation of Compound 9

14-hydroxy angolosamine erythronolide B (9) was generated by feedingcompound 3 (see example 2) to a fermentation of S. rochei ATCC 21250,which adds the hydroxyl group. Compound 9 was then isolated from thefermentation broth using standard methods.

Example 9—Preparation of Compound 10

Compound 6 (6.0 mg, 0.01 mmol) was dissolved in dichloromethane (1 mL)and acetaldehyde (1.0 μL, 0.02 mmol) was added. The reaction was stirredat room temperature and sodium triacetoxyborohydride (2.1 mg, 0.01 mmol)was added. The reaction was stirred for 30 minutes and then quenched bythe addition of concentrated aqueous sodium bicarbonate (25 mL). Theaqueous extract was extracted with ethyl acetate (3×25 mL). The organicextracts were combined, washed with concentrated brine solution and thesolvent was removed in vacuo. The target compound 10 was then purifiedby preparative HPLC.

Example 10—Preparation of Compound 12

Compound 3 (see example 2) was biotransformed to remove both methylgroups from the aminosugar by adding it to a fermentation of ATCC 31771and compound 11 was isolated from the fermentation broth using standardmethods.

Compound 11 is dissolved in THF and acetaldehyde is added. The reactionis stirred at room temperature and sodium cyanoborohydride is added. Thereaction is stirred further and the reaction is quenched by the additionof aqueous sodium bicarbonate. The aqueous extract is extracted withEtOAc (3×vol equivalent). The organic extracts are combined, washed withbrine and the solvent is removed in vacuo. The target compound 12 isthen purified using standard methods.

Example 11—Preparation of Compound 14

Compound 1 (see example 1) is biotransformed to remove a methyl groupfrom the aminosugar by adding it to a fermentation of ATCC 31771 andcompound 13 is isolated from the fermentation broth using standardmethods.

Compound 13 is dissolved in THF and acetaldehyde is added. The reactionis stirred at room temperature and sodium cyanoborohydride is added. Thereaction is stirred further and the reaction is quenched by the additionof aqueous sodium bicarbonate. The aqueous extract is extracted withEtOAc (3×vol equivalent). The organic extracts are combined, washed withbrine and the solvent is removed in vacuo. The target compound 14 isthen purified using standard methods.

Example 12—Preparation of Compound 16

Compound 1 (see example 1) is biotransformed to remove both methylgroups from the aminosugar by adding it to a fermentation of ATCC 31771and compound 15 is isolated from the fermentation broth using standardmethods.

Compound 15 is dissolved in THF and acetaldehyde is added. The reactionis stirred at room temperature and sodium cyanoborohydride is added. Thereaction is stirred further and the reaction is quenched by the additionof aqueous sodium bicarbonate. The aqueous extract is extracted withEtOAc (3×vol equivalent). The organic extracts are combined, washed withbrine and the solvent is removed in vacuo. The target compound 16 isthen purified using standard methods.

Example 13—Assessment of Direct Antibacterial Activity

The bioactivity of macrolide compounds against 4 strains of common gutbacteria (Escherichia coli, Streptococcus salivarius subsp. salivarius,Lactobacillus casei and Bifidobacterium longum subsp. infantis) andcommon mammalian skin isolate Micrococcus luteus, was assessed using theMinimum Inhibitory Concentration (MIC) assay. Bacterial strains werepurchased from DSMZ (Brunswick, Germany) except M. luteus which wasobtained from NCIMB, and stored in 20% glycerol at −80° C.

Stock solutions (100% DMSO) of positive controls (azithromycin anderythromycin), and of test compounds 1 and 2 were diluted in broth toworking stock concentrations of 256 μg/ml (final assay testingconcentration range 128 μg/ml to 0.00391 μg/ml). Stock solutions of allother compounds were diluted in broth to working stock concentrations of128 μg/ml (final assay testing concentration range 64 μg/ml to 0.00195μg/ml). Bacterial strains were cultivated in appropriate broth in ananaerobic chamber at 37° C., except for M. luteus which was incubatedaerobically at 37° C. 18 h cultures were diluted in broth to an OD₅₉₅ of0.1 and then further diluted 1:10. In 96-well plates, in duplicate, 200μl working stock of test compound was transferred to well 1 and seriallydiluted (1:2) in broth. 100 μl bacterial suspension was aliquoted intoeach well and mixed thoroughly. Appropriate sterility controls wereincluded and plates were incubated in an anaerobic chamber, oraerobically (M. luteus) at 37° C. for 18 h. The MIC was determined to bethe concentration of test compound in the first well with no visiblegrowth.

TABLE 1 Escherichia Streptococcus Lactobacillus BifidobacteriumMicrococcus coli salivarius casei longum luteus Azithromycin <8 μg/ml<0.5 μg/ml <1.0 μg/ml >64 μg/ml 0.125 μg/ml Erythromycin >64 μg/ml <0.06μg/ml <0.25 μg/ml >64 μg/ml <0.0625 μg/ml Compound 1 >64 μg/ml >64μg/ml >64 μg/ml >64 μg/ml >256 μg/ml Compound 4 >64 μg/ml >64 μg/ml >64μg/ml >64 μg/ml Compound 5 >64 μg/ml >64 μg/ml >64 μg/ml >64 μg/mlCompound 6 >64 μg/ml >64 μg/ml >64 μg/ml >64 μg/ml Compound 7 >64μg/ml >64 μg/ml >64 μg/ml >64 μg/ml Compound 8 >64 μg/ml >64 μg/ml >64μg/ml >64 μg/ml Compound 9 >64 μg/ml >64 μg/ml >64 μg/ml >64 μg/ml EM70364-128 μg/ml

As can be seen from the data presented in Table 1, compounds 1, 3, 4, 5,6, 7, 8 and 9 show no antibacterial activity against any of thebacterial strains tested, whilst erythromycin and azithromycin showpotent activity against a number of the strains.

Example 14—Assessment of Immune Stimulatory Activity

Human peripheral blood mononuclear cells (PBMCs) were purified fromhealthy donors with Ficoll-Paque density centrifugation. Cells werecultured in complete RPMI-1640 medium (Invitrogen) supplemented with 25mM HEPES, L-glutamine, Sodium pyruvate (Sigma), 10% fetal bovine serum,100 μg/mL penicillin and 100 μg/mL streptomycin (Hyclone). Cells werestimulated for 24 h (study 1-4) or 48 h to 1 week (study 5) in 37° C.,5% CO₂ with increasing concentrations of compound 1 and 2 in tissueculture plates. The cells were removed from the plate, washed in PBS andanalysed for expression of cell specific surface markers and MHC class Iwith flow cytometry using monoclonal antibodies from BD Pharmingen and aFACS Canto II flow cytometer.

Supernatant IL-10 was measured with a standard sandwich ELISA (allantibodies from BD Biosciences) after 48 hours and 7 days incubationwith 2.5 uM of compound 1 and 100 U/mL IL-2 (Miltenyi Biotechnologies)in complete RPMI media, 37° C., 5% CO₂.

Study 1: After 24 h of in vitro stimulation of peripheral bloodmononuclear cells (PBMC) with 1 μM compound 1 (FIG. 8) the activationmarker CD69 was upregulated on CD4+ T cells and B cells (FIG. 1).

Study 2: We also observed upregulation of the molecule MHC class I(HLA-ABC) on T- and B-cells (FIG. 2), indicating an effect on antigenpresentation of viral antigens.

Study 3: Stimulation of PBMC with compound 1 led to the upregulation ofthe co-stimulatory molecule CD80 as well as the antigen presentingmolecule MHC class II (HLA-DR) on monocytes (FIG. 3).

Study 4: Monocytes differentiated into macrophages also upregulated CD80in response to stimulation by compound 1 (FIG. 4).

Study 5: PBMCs stimulated with compound 1 for 48 h and 7 days expressedan altered cytokine profile with increased production of theimmunosuppressive cytokine IL-10, measured with sandwich ELISA. Thisindicate an immune inhibitory effect under certain conditions (FIG. 5).

Study 6: PBMC were stimulated with compound 1 and cultured in RPMI mediafor 6 days in the presence of IL-2 (Miltenyi Biotechnologies) and CellTrace Violet Dye (Invitrogen). Proliferation was measured with flowcytometry. Analysis of the immunological effect of compound 1 revealedan altered cytokine driven proliferation profile of T cells (FIG. 6).

Study 7: CMV virus specific T cell proliferation was also affected bycompound 1. PBMCs from cytomegalovirus (CMV) infected donors cultured inthe presence of CMV antigen and compound 1 for 6 days displayed analtered phenotype of activated CMV specific CD8+ T cells with anincreased expression of IL-7 receptor α (CD127), measured with flowcytometry (FIG. 7). CD127 is crucial for T cell homeostasis,differentiation and function, and reduced expression correlates withdisease severity in HIV and other chronic viral diseases (Crawley et al.2012).

As can be seen, compound 1 has a surprising ability to specificallyactivate and modify an immune response by affecting antigenpresentation, co-stimulation and T cell activation and proliferation. Inmany of these studies, compound 2, another related macrolideerythromycin analogue with altered glycosylation, previously publishedin Schell et al, 2008 (as compound 20), was included and showed littleor no activity in the assays.

Study 8: PBMCs from CMV infected donors cultured in the presence of CMVantigen where either untreated or exposed to compound 1 or compound 2for 3 days. Exposure to compound 1 induced secretion of high levels ofIFN-gamma, whereas antigen culture alone or antigen together withcompounds 2 did not induce IFN-gamma secretion (FIG. 9).

Study 9: Macrophages from healthy donors where exposed to compounds 1 or2 for 48 hours. Only macrophages exposed to compound 1 secretedIFN-gamma whereas untreated macrophages and macrophages exposed tocompound A did not secrete IFN-gamma (FIG. 10). Compound 1 is thereforeable to induce IFN-gamma secretion in macrophages from healthy donors.

Study 10: PBMCs and macrophages where exposed to compounds 1 or 2 for 2days (FIG. 11). Basal expression of RANTES in PBMCs was unaffected bycompound 2, whereas compound 1 induced a twofold upregulation ofexpression. Expression of RANTES was miniscule in macrophages, andcompound 1 induced a high expression.

Study 11: PBMCs and macrophages where exposed to compounds 1 and 2 for 2days. PBMCs and macrophages secreted IL-12p70 in response to compound 1,whereas compound 2 failed to induce secretion over untreated cells (FIG.12).

Study 12: PBMCs, macrophages and CD4+ T cells where exposed to compounds1 and 2 for 2 days. IL-1beta secretion was increased by compound 1 inmacrophages and slightly in PBMCs while no IL-1beta was induced in CD4+T cells (FIG. 13).

Study 13: Compound 1 was administered i.v. to C57bl/6 mice at 0.165mg/kg to 5 mg/kg. CD25+ cell abundance was increased in animalsreceiving the highest dose of 5 mg/kg (FIG. 14), as was body weight inthe same group (not shown).

Study 14: Compound 1 or A was administered i.v. to C57bl/6 mice. 24 hlater the spleen was removed and MHC class I expression on CD11 b+splenocytes was assessed Compound 1 induced an increase in splenocytecells with high MHC I expression, whereas no effect was observed insplenocytes from mice injected with compound A.

Example 15—Assessment of Metabolic Stability

The metabolic stability of the compounds of the invention was assessedin a standard human microsome stability assay (see general methods).Compounds with longer half-lives would be expected to have longerhalf-lives following dosing, which can be useful to allow less frequentdosing. Compounds with shorter half-lives could be useful for use as‘soft drugs’ where the active entity degrades rapidly once entering thepatient's system. The half-life of the compounds assessed in shown intable 2 below:

TABLE 2 T1/2 (minutes) Azithromycin 245 Erythromycin 31 Compound 1 108Compound 3 35 Compound 4 160 Compound 5 83 Compound 6 109 Compound 7 56Compound 8 33 Compound 9 100 Compound 10 31 Compound 17 151 Compound 1825 Compound 19 18 EM703 97

As can be seen, many of the compounds of the invention have increased ordecreased metabolic stability as compared to azithromycin, erythromycinand EM703 (e.g. see EP1350510).

Example 16—Assessment of Caco-2 Permeability

The permeability of the compounds of the invention was assessed in astandard caco-2 bidirectional permeability assay (see general methods).Compounds with increased permeability would be expected to have bettercell penetration and potential for effect, those with improvedpermeability and/or reduced efflux would be expected to have increasedoral bioavailability. The permeability and efflux of the compounds isshown in table 3 below:

TABLE 3 P_(app) × 10⁶/cm.s⁻¹ Efflux ratio Azithromycin <0.14 >78Compound 1 0.32 63 Compound 3 0.27 166 Compound 4 0.38 49 Compound 50.47 81 Compound 8 0.46 56 Compound 10 1.23 26 Compound 17 0.5 39Compound 18 9.44 3.5 EM703 <0.15 >108

As can be seen, many of the compounds of the invention have improvedcell permeability and/or reduced efflux as compared to azithromycin andEM703 (e.g. see EP1350510).

Example 17—Assessment of TLR2 Stimulation

Compounds were tested using a TLR2 reporter assay (see general methods)that measured for stimulation of the TLR2 receptor. Stimulatory effectwas measured as an increase in optical density (OD) due to release ofsecreted alkaline phosphatase (SEAP) and is shown in table 4:

TABLE 4 OD after addition OD after addition OD after addition of 20 μMtest of 10 μM test of 5 μM test article article article Azithromycin0.031 0.045 0.029 Erythromycin 0.045 0.065 0.035 Compound 1 0.458 0.2020.111 Compound 2 0.044 0.010 0.046 Compound 3 −0.026 −0.015 −0.043Compound 17 0.234 0.155 0.054 EM703 −0.033 −0.024 −0.040

As can be seen, compound 1 stimulated TLR2 at concentrations down to 5uM, compound 17 stimulated TLR2 at concentrations down to 10 uM, whilsterythromycin A, azithromycin and compounds 2 and 3, related macrolideerythromycin analogues with altered glycosylation, previously publishedin Schell et al, 2008 (as compounds 17 and 20), showed little or nostimulation at concentrations up to 20 uM.

Example 18—Preparation of Compound 17

The aglycone 17a was generated from9-deoxo-8a-aza-8a-methyl-8a-homoerythromycin (Wilkening 1993) followedby hydrolysis of the sugars. 17a was then fed to a biotransformationstrain capable of adding angolosamine to the 3-hydroxyl (such as NCIMB42718) and compound 17 isolated from the fermentation broth usingstandard methods.

Example 19—Preparation of Compound 18

6-deoxy erythronolide B (6-DEB, 18a) was fed to a biotransformationstrain capable of adding angolosamine to the 3-hydroxyl (such as NCIMB42718) and isolated from the fermentation broth using standard methods.

REFERENCES

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All references referred to in this application, including patent andpatent applications, are incorporated herein by reference to the fullestextent possible.

Specific embodiments of the invention are given in the following list ofitems.

Item 1

A compound of Formula I

wherein X is selected from C═O, —NR₃CH₂—, —CH₂NR₃—, —NR₃(C═O)—,—(C═O)NR₃—, C═NOH, and —CH(OH)—, and R₂ is a sugar of Formula (II) orFormula (III):

wherein R₁ is selected from an alkyl, heteroalkyl, cycloalkyl, aryl, andheteroaryl moiety,wherein heteroatoms are selected from O, N, P, and S,wherein alkyl moiety is selected from C₁-C₆ alkyl groups that areoptionally branched,wherein heteroalkyl moiety is selected from C₁-C₆ alkyl groups that areoptionally branched or substituted and that optionally comprise one ormore heteroatoms,wherein cycloalkyl moiety is selected from a C₁-C₆ cyclic alkyl groupsthat are optionally substituted and that optionally comprise one or moreheteroatoms,wherein aryl moiety is selected from optionally substituted C₆ aromaticrings,wherein heteroaryl moiety is selected from optionally substituted C₁-C₅aromatic rings comprising one or more heteroatoms,wherein substituents, independently, are selected from alkyl, OH, F, Cl,NH₂, NH-alkyl, NH-acyl, S-alkyl, S-acyl, O-alkyl, and O-acyl,wherein acyl is selected from C₁-C₄ optionally branched acyl groups,wherein R₃ is selected from H and Me,wherein R₄ is selected from H and Me,wherein R_(a) is selected from H and CR₂₁R₂₂R₂₃,wherein R₂₁, R₂₂, R₂₃, and R₅, R₆, R₇, R₈, R₉, and R₁₀, independently,are selected from H, Me, NR₁₁R₁₂, NO₂, and OR₁₁,wherein R₂₃ together with R₄ in Formula (II), R₄ together with R₅ inFormula (II), R₅ together with R₇ in Formula (II), and R₇ together withR₉ in Formula (II), independently, may be joined to represent a bond toleave a double bond between the carbon atoms that each group isconnected to,wherein R₂₁ together with R₂₂, R₅ together with R₆, R₇ together with R₈,or R₉ together with R₁₀ may be replaced with a carbonyl,wherein R₁₁ and R₁₂, independently, are selected from H and alkyl,wherein R₁₃ is selected from H, OH, and OCH₃,wherein R₁₄ is selected from H and OH,and wherein one of R₅, R₆, R₇, R₈, R₉ or R₁₀ is selected from NR₁₁R₁₂and NO₂,with the proviso that when R₁ is Et, R₂ is a sugar of Formula (II), R₁₃is OH, R₁₄ is H, R_(a) is H, R₄ is Me, R₅ is H, R₆ is OH, R₇ is H, R₈ isNR₁₁R₁₂, R₉ is H, and R₁₀ is H, X may not be C═O.

Item 2

A compound of item 1

with the proviso that when R₁ is Et, R₂ is a sugar of Formula (II), R₁₃is H or OH, R₁₄ is H or OH, R_(a) is H, R₄ is Me, R₅ is H, R₆ is OH, R₇is H, R₈ is NR₁₁R₁₂, R₉ is H, and R₁₀ is H, X may not be C═O.

with the proviso that when R₁ is Et, R₂ is a sugar of Formula (II), R₁₃is H or OH, R₁₄ is H or OH, R_(a) is H, R₄ is Me, R₅ is OH, R₆ is H, R₇is OH, R₈ is Me, R₉ is H, and R₁₀ is H, X may not be C═O.

with the proviso that when R₁ is Et, R₂ is a sugar of Formula (II), R₁₃is H or OH, R₁₄ is H or OH, R_(a) is H, R₄ is Me, R₅ is OH, R₆ is H, R₇is H, R₈ is NR₁₁R₁₂, R₉ is H, and R₁₀ is OH, X may not be C═O.

Item 3

A compound according to Item 1 or 2,

wherein R₂₃ together with R₄ in Formula (II), R₄ together with R₅ inFormula (II), R₅ together with R₇ in Formula (II), and R₇ together withR₉ in Formula (II), independently, may be joined to represent a bond toleave a double bond between the carbon atoms that each group isconnected to, so thatwherein if R₂₃ and R₄ are joined to form a double bond, then Formula(II) can be represented by:

wherein if R₄ and R₅ are joined to form a double bond, then Formula (II)can be represented by:

wherein if R₅ and R₇ are joined to form a double bond, then Formula (II)can be represented by:

wherein if R₇ and R₉ are joined to form a double bond, then Formula (II)can be represented by:

wherein R₄ together with R₅ in Formula (III), R₄ together with R₇ inFormula (III), and R₇ together with R₉ in Formula (III), independently,may be joined to represent a bond to leave a double bond between thecarbon atoms that each group is connected to, so thatwherein if R₄ and R₅ are joined to form a double bond, then Formula(III) can be represented by:

wherein if R₄ and R₇ are joined to form a double bond, then Formula(III) can be represented by:

wherein if R₇ and R₉ are joined to form a double bond, then Formula(III) can be represented by:

Item 4

A compound according to Formula I

wherein X is selected from C═O, —NR₃CH₂—, and —CH(OH)—, and R₂ is asugar of Formula (II):

wherein R₁ is selected from and alkyl or cycloalkyl moiety,wherein alkyl moiety is selected from C₁-C₆ alkyl groups that areoptionally branched and, independently, optionally hydroxylated,wherein cycloalkyl moiety is selected from C₁-C₆ optionally substitutedcyclic alkyl groups,wherein substituents are selected from alkyl and OH,wherein R₃ is selected from H and Me,wherein R₄ is selected from H and Me,wherein R_(a) is selected from H and CR₂₁R₂₂R₂₃,wherein R₂₁, R₂₂, R₂₃, and R₅, R₆, R₇, R₈, R₉, and R₁₀, independently,are selected from H, Me, NR₁₁R₁₂, NO₂, and OR₁₁,wherein R₂₃ together with R₄ in Formula (II), R₄ together with R₅ inFormula (II), R₅ together with R₇ in Formula (II), and R₇ together withR₉ in Formula (II), independently, may be joined to represent a bond toleave a double bond between the carbon atoms that each group isconnected to, so thatwherein if R₂₃ and R₄ are joined to form a double bond, then Formula(II) can be represented by:

wherein if R₄ and R₅ are joined to form a double bond, then Formula (II)can be represented by:

wherein if R₅ and R₇ are joined to form a double bond, then Formula (II)can be represented by:

wherein if R₇ and R₉ are joined to form a double bond, then Formula (II)can be represented by:

wherein R₂₁ together with R₂₂, R₅ together with R₆, R₇ together with R₈,or R₉ together with R₁₀ may be replaced with a carbonyl,wherein R₁₁ and R₁₂, independently, are selected from H and alkyl,wherein R₁₃ is selected from H, OH, and OCH₃,wherein R₁₄ is selected from H and OH,and wherein one of R₅, R₆, R₇, R₈, R₉ or R₁₀ is selected from NR₁₁R₁₂and NO₂,with the proviso that when R₁ is Et, R₂ is a sugar of Formula (II), R₁₃is OH, R₁₄ is H, R_(a) is H, R₄ is Me, R₅ is H, R₆ is OH, R₇ is H, R₈ isNR₁₁R₁₂, R₉ is H, and R₁₀ is H, X may not be C═O.

Item 5

A compound according to any one of the preceding items, wherein R₂ isselected from L-daunosamine, L-acosamine, L-ristosamine, D-ristosamine,4-oxo-L-vancosamine, L-vancosamine, D-forosamine, L-actinosamine,3-epi-L-vancosamine, L-vicenisamine, L-mycosamine, D-mycosamine,D-3-N-methyl-4-O-methyl-L-ristosamine, D-desosamine,N,N-dimethyl-L-pyrrolosamine, L-megosamine, L-nogalamine, L-rhodosamine,D-angolosamine, L-kedarosamine, 2′-N-methyl-D-fucosamine,3-N,N-dimethyl-L-eremosamine, D-ravidosamine,3-N,N-dimethyl-D-mycosamine/D-mycaminose, 3-N-acetyl-D-ravidosamine,4-O-acetyl-D-ravidosamine, 3-N-acetyl-4-O-acetyl-D-ravidosamine,D-glucosamine, N-acetyl-D-glucosamine, L-desosamine, D-amosamine,D-viosamine, L-avidinosamine, D-gulosamine, D-allosamine, andL-sibirosamine.

Item 6

A compound according to any one of the preceding items, wherein R₂ isselected from D-angolosamine, N-desmethyl D-angolosamine, N-didesmethylD-angolosamine, N-desmethyl N-ethyl D-angolosamine, and N-didesmethylN-diethyl D-angolosamine.

Item 7

A compound according to any one of the preceding items, wherein R₂ is asugar according to Formula (II).

Item 8

A compound according to any one of the preceding items, wherein R₂ is asugar according to formula II wherein R_(a) is H, R₄ is Me, R₅ is H, R₆is OH, R₇ is H, R₈ is NR₁₁R₁₂, R₉ is H and R₁₀ is H.

Item 9

A compound according to any one of the preceding items, wherein R₁₁ isselected from H, Me, and Et, and R₁₂ is selected from H, Me, and Et.

Item 10

A compound according to any one of the preceding items, wherein R₁₁ isEt and R₁₂ is Et.

Item 11

A compound according to any one of items 1-8, wherein R₁₁ is Me and R₁₂is Et.

Item 12

A compound according to any one of the preceding items, wherein X isselected from C═O, —NR₃CH₂— and —CH(OH)—

Item 13

A compound according to any one of the preceding items, wherein R₁ isselected from Me, Et, and cycloalkyl.

Item 14

A compound according to any one of the preceding items, wherein R₁ isselected from Me and Et and cycloalkyl.

Item 15

A compound according to any one of the preceding items selected from:

Item 16

A compound as defined in any one of the preceding items for use inmedicine.

Item 17

A compound as defined in any one of items 1-15 for use in the treatmentof intracellular infections.

Item 18

A compound according for use according to item 17, wherein theintracellular infection is selected from bacterial, protozoal, andfungal infections.

Item 19

A compound for use as defined in item 18, wherein the intracellularinfection is selected from bacterial infections caused by Mycobacteriumtuberculosis, Mycobacteria causing atypical disease, Mycobacterium aviumand M. intracellulare (also known as Mycobacterium avium-intracellularecomplex, or MAC), M. kansasii, M. marinum, M. fortuitum, M. gordinae,Mycoplasma pneumoniae, M. genitalium, M. hominis, Ureaplasmaurealyticum, U. parvum, Chlamydophila pneumoniae, and Salmonellatyphimurium, protozoal infections caused by Toxoplasma gondii,Plasmodium falciparum, P. vivax, Trypanosoma cruzi, Cryptosporidium, andLeishmania, and fungal infections caused by Histoplasma capsulatum,Cryptococcus neoformans, and Encephalitozoon cuniculi.

Item 20

A compound for use as defined in item 18, wherein the intracellularinfection is selected from bacterial infections caused by Mycobacteriumtuberculosis, Mycobacteria causing atypical disease, Mycobacterium aviumand M. intracellulare (also known as Mycobacterium avium-intracellularecomplex, or MAC), M. kansasii, M. marinum, M. fortuitum, M. gordinae,Mycoplasma pneumoniae, M. genitalium, M. hominis, Ureaplasmaurealyticum, U. parvum, Chlamydophila pneumoniae, and Salmonellatyphimurium.

Item 21

A compound for use as defined in item 18, wherein the intracellularinfection is selected from protozoal infections caused by Toxoplasmagondii, Plasmodium falcipa-rum, P. vivax, Trypanosoma cruzi,Cryptosporidium, and Leishmania.

Item 22

A compound for use as defined in item 18, wherein the intracellularinfection is selected from fungal infections caused by Histoplasmacapsulatum, Cryptococcus neoformans, and Encephalitozoon cuniculi.

Item 23

A method for preparing a compound as defined in any one of items 1-15,the method comprising addition of an aglycone with Formula (IV)

to a culture of a biotransformation strain which glycosylates at the3-hydroxyl position.

Item 24

A method according to item 24, wherein the biotransformation strainexpresses glycosyltransferases with >70% homology to AngMII and AngMIII

The invention claimed is:
 1. A compound of Formula (I), wherein thecompound has the following structure:

wherein: X is selected from —NR₃CH₂—, —CH₂NR₃—, —NR₃(C═O)—, —(C═O)NR₃—,and C═NOH; R2 is a sugar of Formula (II) or Formula (III):

R₁ is selected from an alkyl, heteroalkyl, cycloalkyl, aryl, andheteroaryl moiety, wherein the alkyl moiety is selected from C₁-C₆ alkylgroups that are optionally branched, the heteroalkyl moiety is selectedfrom C₁-C₆ alkyl groups that are optionally branched or substituted andthat optionally comprise one or more heteroatoms, the cycloalkyl moietyis selected from a C₁-C₆ cyclic alkyl groups that are optionallysubstituted and that optionally comprise one or more heteroatoms, thearyl moiety is selected from optionally substituted C₆ aromatic rings,the heteroaryl moiety is selected from optionally substituted C₁-C₅aromatic rings comprising one or more heteroatoms, wherein the one ormore heteroatoms are selected from O, N, P, and S, and the heteroalkyl,cycloalkyl, aryl, and heteroaryl moieties, when substituted,independently, are substituted with one or more groups selected fromalkyl, OH, F, Cl, NH₂, NH-alkyl, NH-acyl, S-alkyl, S-acyl, O-alkyl, andO-acyl, wherein acyl is selected from C₁-C₄ optionally branched acylgroups; R3 is selected from H and Me; R4 is selected from H and Me;R_(a) is selected from H and —CR₂₁R₂₂R₂₃; R₂₁, R₂₂, R₂₃, and R₅, R₆, R₇,R₈, R₉, and R₁₀, independently, are selected from H, Me, NR₁₁R₁₂, NO₂,and OR₁₁, wherein R₂₃ together with R₄ in Formula (II), R4 together withR₅ in Formula (II), R5 together with R₇ in Formula (II), and R₇ togetherwith R₉ in Formula (II), independently, may be joined to represent abond to leave a double bond between the carbon atoms that each group isconnected to, and R₂₁ together with R₂₂, R₅ together with R₆, R₇together with R₈, or R₉ together with R₁₀ may be replaced with acarbonyl; R₁₁ and R₁₂, independently, are selected from H and alkyl; R₁₃is selected from H, OH, and OCH₃; R₁₄ is selected from H and OH; and oneof R₅, R₆, R₇, R₈, R₉, or R₁₀ is selected from NR₁₁R₁₂ and NO₂; or apharmaceutically acceptable salt, hydrate, solvate, tautomer, enantiomeror diastereomer thereof.
 2. The compound according to claim 1, wherein:X is selected from —NR₃CH₂— and —CH₂NR₃; and R₂ is a sugar of Formula(II).
 3. The compound according to claim 1, wherein R₁ is methyl orethyl.
 4. The compound according to claim 1, wherein one of R₅, R₆, R₇,or R₈ is NR₁₁R₁₂.
 5. The compound according to claim 1, wherein R₂₁,R₂₂, R₂₃, and R₅, R₆, R₇, R₈, R₉, and R₁₀, independently, are selectedfrom H, Me, NR₁₁R₁₂, and OR₁₁.
 6. The compound according to claim 1,wherein R₁₃ and R₁₄ are OH.
 7. The compound according to claim 1,wherein X is selected from —NR₃CH₂— and —CH₂NR₃—; R₂ is a sugar ofFormula (II); R₁ is methyl or ethyl; R₃ is selected from H and Me; R₄ isH; R_(a) is —CR₂₁R₂₂R₂₃; R₂₁, R₂₂, R₂₃, and R₅, R₆, R₇, R₈, R₉, and R₁₀,independently, are selected from H, Me, NR₁₁R₁₂, NO₂, and OR₁₁; R₁₁, andR₁₂, independently, are selected from H and an alkyl moiety, wherein thealkyl moiety is selected from C₁-C₆ alkyl groups that are optionallybranched; R₁₃ is selected from H, OH, and OCH₃; R₁₄ is selected from Hand OH; and one of R₅, R₆, R₇, R₈, R₉, or R₁₀ is NR₁₁R₁₂.
 8. Thecompound according to claim 1, wherein R₂ is a sugar according toFormula (II), R_(a) is H, R₄ is Me, R₅ is H, R₆ is OH, R₇ is H, R₈ isNR₁₁R₁₂, R₉ is H, and R₁₀ is H.
 9. The compound according to claim 1,wherein R₁₁, and R₁₂, independently, are selected from H, Me, and Et.10. The compound according to claim 1, wherein X is —NR₃CH₂—.
 11. Thecompound according to claim 1, wherein R₁ is Et.
 12. A compound selectedfrom:

or a pharmaceutically acceptable salt thereof.
 13. The compoundaccording to claim 12, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 14. A pharmaceuticalcomposition comprising the compound of claim
 1. 15. A method fortreating an intracellular infection, the method comprising administeringto a subject in need thereof a therapeutically effective amount of thecompound of claim
 1. 16. The method of claim 15, wherein theintracellular infection is selected from bacterial infections caused byMycobacterium tuberculosis, Mycobacteria causing atypical disease,Mycobacterium avium and M. intracellulare (also known as Mycobacteriumavium-intracellulare complex, or MAC), M. kansasii, M. marinum, M.fortuitum, M. gordinae, Mycoplasma pneumoniae, M. genitalium, M.hominis, Ureaplasma urealyticum, U. parvum, Chlamydophila pneumoniae,and Salmonella typhimurium.
 17. The method of claim 15, wherein theintracellular infection is selected from protozoal infections caused byToxoplasma gondii, Plasmodium falciparum, P. vivax, Trypanosoma cruzi,Cryptosporidium, and Leishmania.
 18. The method of claim 15, wherein theintracellular infection is selected from fungal infections caused byHistoplasma capsulatum, Cryptococcus neoformans, and Encephalitozooncuniculi.
 19. A method for preparing a compound according to claim 1,the method comprising adding an aglycone of Formula (IV):

to a culture of a biotransformation strain which glycosylates at the3-hydroxyl position of a compound of Formula (IV), wherein thebiotransformation strain expresses glycosyltransferases with 70% or morehomology to AngMII (SEQ ID NO: 1) or AngMIII (SEQ ID NO: 2).